HDAC inhibitors for the treatment of diabetic peripheral neuropathy

ABSTRACT

The invention relates to HDAC inhibitors for use in the treatment of diabetic peripheral neuropathy in a subject in need thereof. Also provided herein are methods for treating diabetic peripheral neuropathy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an HDAC inhibitor.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/335,971, filed on Oct. 27, 2016, which application claims priority toU.S. Patent Application No. 62/246,965 filed Oct. 27, 2015, which claimspriority to U.S. Patent Application No. 62/281,990, filed Jan. 22, 2016,each of which is incorporated herein by reference in its entirety.

BACKGROUND

Diabetic Peripheral Neuropathy (DPN) is a major complication of diabetesand affects 30-50% of diabetic patients. It is characterized byprogressive distal to proximal degeneration of peripheral nerve axons,pain and loss of sensation. The underlying mechanisms of DPN are poorlyunderstood and despite the prevalence and considerable symptom severity,therapeutic intervention options are limited.

Histone deacetylase (HDAC) enzymes represent attractive therapeutictargets in the treatment of diabetic peripheral neuropathy.

SUMMARY

Provided herein are pharmaceutical compounds for use in the treatment ofdiabetic peripheral neuropathy in a subject in need thereof. Alsoprovided herein are methods for treating diabetic peripheral neuropathyin a subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the total distance traveled by a population ofmitochondria under each condition (25 mM glucose (control), 200 mMglucose or 200 mM glucose and 500 nM Compound 2) as described in Example4.

FIG. 1B shows that treatment with Compound 2 restores normal pattern ofmitochondrial movement as described in Example 4.

FIG. 2A shows the mean response to Von Frey test (g) in the study ofdiabetic peripheral neuropathy in STZ diabetic rats upon treatment withCompound 1 as described in Example 5.

FIG. 2B shows the mean response to Von Frey test (g) in the study ofdiabetic peripheral neuropathy in STZ diabetic rats upon treatment withCompound 2 as described in Example 5.

FIG. 2C shows the mean response to Von Frey test (g) in the study ofdiabetic peripheral neuropathy in STZ diabetic rats upon treatment withCompound 3 as described in Example 5.

FIG. 3A shows Compound 2 induces tubulin hyperacetylation in rat dorsalroot ganglion neurons (DRGN) as described in Example 6.

FIG. 3B shows Compound 2 increases acetylation of axonal microtubules asdescribed in Example 6.

FIG. 4 shows the effects of Compound 1 and Compound 2 on latency time inthe Tail Flick test described in Example 7.

FIG. 5A shows changes in acyl-carnitines, AC 16:2 upon administration ofvehicle or Compound 1 as described in Example 8.

FIG. 5B shows changes in acyl-carnitines, AC 18:2 upon administration ofvehicle or Compound 1 as described in Example 8.

DETAILED DESCRIPTION

Provided herein are pharmaceutical compounds for use in the treatment ofdiabetic peripheral neuropathy in a subject in need thereof. Alsoprovided herein are methods for treating diabetic peripheral neuropathyin a subject in need thereof. Specifically, provided herein are HDAC6inhibitors for the treatment of diabetic peripheral neuropathy.

Thus, in an aspect, provided herein is a method for treating orpreventing diabetic peripheral neuropathy in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of an HDAC6 inhibitor.

In an embodiment, the HDAC6 inhibitor is an HDAC6-specific inhibitor.

The compounds provided herein (e.g., Compound 1 and Compound 2) areeffective at reversing tacticle allodynia in a rat model of diabeticneuropathy (see Example 5). Further, HDAC-6 specific inhibitor, Compound1, has shown disease modifying effects in the diabetic neuropathy model(Example 5). This result indicates that HDAC6 is a promising target forthe treatment of peripheral neuropathies. Further, without being boundby a theory, it is suggested that HDAC6 inhibitors exert their effectsat least partially by restoring normal axonal transport in a diabeticneuropathy setting (see, e.g., Example 4).

Definitions

Listed below are definitions of various terms used herein. Thesedefinitions apply to the terms as they are used throughout thisspecification and claims, unless otherwise limited in specificinstances, either individually or as part of a larger group.

The term “about” generally indicates a possible variation of no morethan 10%, 5%, or 1% of a value. For example, “about 25 mg/kg” willgenerally indicate, in its broadest sense, a value of 22.5-27.5 mg/kg,i.e., 25±2.5 mg/kg.

The term “alkyl” refers to saturated, straight- or branched-chainhydrocarbon moieties containing, in certain embodiments, between one andsix (C₁₋₆ alkyl), or one and eight carbon atoms (C₁₋₈ alkyl),respectively. Examples of C₁₋₆ alkyl moieties include, but are notlimited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl,neopentyl, n-hexyl moieties; and examples of C₁₋₈ alkyl moietiesinclude, but are not limited to, methyl, ethyl, propyl, isopropyl,n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

The number of carbon atoms in an alkyl substituent can be indicated bythe prefix “C_(x-y),” where x is the minimum and y is the maximum numberof carbon atoms in the substituent. Likewise, a C_(x) chain means analkyl chain containing x carbon atoms.

The term “alkoxy” refers to an —O-alkyl moiety.

The term “aryl” refers to a mono- or poly-cyclic carbocyclic ring systemhaving one or more aromatic rings, fused or non-fused, including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyland the like. In some embodiments, aryl groups have six carbon atoms. Insome embodiments, aryl groups have from six to ten carbon atoms(C₆₋₁₀-aryl). In some embodiments, aryl groups have from six to sixteencarbon atoms (C₆₋₁₆-aryl).

The term “heteroaryl” refers to a mono- or poly-cyclic (e.g., bi-, ortri-cyclic or more) fused or non-fused, moieties or ring system havingat least one aromatic ring, having from five to ten ring atoms of whichone ring atom is selected from S, O, N and Si; zero, one or two ringatoms are additional heteroatoms independently selected from S, O, N andSi; and the remaining ring atoms are carbon. Heteroaryl includes, but isnot limited to pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl,benzooxazolyl, quinoxalinyl, and the like.

The term “halo” refers to a halogen, such as fluorine, chlorine,bromine, and iodine.

The term “HDAC” refers to histone deacetylases, which are enzymes thatremove the acetyl groups from the lysine residues in core histones, thusleading to the formation of a condensed and transcriptionally silencedchromatin. There are currently 18 known histone deacetylases, which areclassified into four groups. Class I HDACs, which include HDAC1, HDAC2,HDAC3, and HDAC8, are related to the yeast RPD3 gene. Class II HDACs,which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, are relatedto the yeast Hda1 gene. Class III HDACs, which are also known as thesirtuins are related to the Sir2 gene and include SIRT1-7. Class IVHDACs, which contains only HDAC11, has features of both Class I and IIHDACs. The term “HDAC” refers to any one or more of the 18 known histonedeacetylases, unless otherwise specified.

The term “HDAC6-specific” means that the compound binds to HDAC6 to asubstantially greater extent, such as 4×, 5×, 10×, 15×, 20× greater ormore, than to any other type of HDAC enzyme, such as HDAC1 or HDAC2.That is, the compound is selective for HDAC6 over any other type of HDACenzyme. For example, a compound that binds to HDAC6 with an IC₅₀ of 10nM and to HDAC1 with an IC₅₀ of 50 nM is HDAC6-specific. On the otherhand, a compound that binds to HDAC6 with an IC₅₀ of 50 nM and to HDAC1with an IC₅₀ of 60 nM is not HDAC6-specific.

The term “inhibitor” is synonymous with the term antagonist.

The term “pharmaceutically acceptable salt” refers to those salts of thecompounds formed by the process of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Additionally, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present invention can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal ofPharmaceutical Science, 66, 2 (1977), each of which is incorporatedherein by reference in its entirety.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable” refers to compounds which possess stability sufficient toallow manufacture and which maintains the integrity of the compound fora sufficient period of time to be useful for the purposes detailedherein (e.g., therapeutic or prophylactic administration to a subject).

The term “subject” refers to a mammal. A subject therefore refers to,for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like.Preferably the subject is a human. When the subject is a human, thesubject may be referred to herein as a patient.

The term “treating” or “treatment” as used herein comprises a treatmentrelieving, reducing or alleviating at least one symptom in a subject oreffecting a delay of progression of a disease. For example, treatmentcan be the diminishment of one or several symptoms of a disorder orcomplete eradication of a disorder, such as the diabetic peripheralneuropathy. Within the meaning of the present disclosure, the term“treat” also denotes to arrest, delay the onset (i.e., the period priorto clinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. The term “protect” is used herein tomean prevent delay or treat, or all, as appropriate, development orcontinuance or aggravation of a disease in a subject, e.g., a mammal orhuman. The term “prevent,” “preventing,” or “prevention,” as used hereincomprises the prevention of at least one symptom associated with orcaused by the state, disease or disorder being prevented.

Histone Deacetylase (HDAC) Inhibitors

In an aspect, provided herein is a method for treating or preventingdiabetic peripheral neuropathy in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of anHDAC6 inhibitor.

In an embodiment, the HDAC6 inhibitor is an HDAC6-specific inhibitor.

In an embodiment, the HDAC6 inhibitor is a compound of Formula Ia:

or a pharmaceutically acceptable salt thereof,

wherein,

R_(x) is selected from H and C₁₋₆-alkyl;

R_(y) is selected from H and C₁₋₆-alkyl;

or R_(x) and R_(y) together with the carbon to which each is attached,form a cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, orcyclooctyl;

each R_(A) is independently C₁₋₆-alkyl, C₁₋₆-alkoxy, halo, OH, orhaloalkyl; and

m is 0, 1, or 2.

In an embodiment, the compound of Formula Ia is selected from thefollowing:

or pharmaceutically acceptable salts thereof.

In an embodiment, the compound of Formula Ia is selected from thefollowing:

or pharmaceutically acceptable salts thereof.

In an embodiment, the HDAC6 inhibitor provided herein is a compound ofFormula I:

or a pharmaceutically acceptable salt thereof,

wherein,

R_(x) and R_(y) together with the carbon to which each is attached, forma cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, orcyclooctyl;

each R_(A) is independently C₁₋₆-alkyl, C₁₋₆-alkoxy, halo, OH, orhaloalkyl; and

m is 0, 1, or 2.

In an embodiment, the compound of Formula I is selected from thefollowing:

or pharmaceutically acceptable salts thereof.

Although the compounds of Formulae Ia and I are depicted in theirneutral forms, in some embodiments, these compounds are used in apharmaceutically acceptable salt form.

In a further embodiment, the compound of Formula I is an HDAC6-specificinhibitor, i.e., the compound of Formula I has a selectivity for HDAC6when tested in an HDAC enzyme assay of about 5 to 1000 fold greater thanfor other HDACs.

The preparation and properties of certain HDAC6 specific inhibitorsaccording to Formula Ia and I are provided in International PatentApplication No. PCT/US2011/060791, the entire content of which isincorporated herein by reference in its entirety.

In another embodiment, the HDAC6 inhibitor provided herein is a compoundof Formula II:

or a pharmaceutically acceptable salt thereof,

wherein ring B is aryl or heteroaryl optionally substituted by OH, halo,or C₁₋₆-alkyl;

R₁ is aryl or heteroaryl optionally substituted by OH, halo, orC₁₋₆-alkyl; and

R is H or C₁₋₆-alkyl.

In an embodiment, the compound of Formula II is selected from thefollowing:

Although the compounds of Formula II are depicted in their neutralforms, in some embodiments, these compounds are used in apharmaceutically acceptable salt form.

In a further embodiment, the compound of Formula II is an HDAC6-specificinhibitor, i.e., it has a selectivity for HDAC6 when tested in an HDACenzyme assay of about 5 to 1000 fold greater than for other HDACs.

The syntheses of compounds of Formula II (e.g., Compound 2) are providedin PCT/US2011/021982; the content of this application is incorporatedherein by reference in its entirety.

In an aspect, provided herein is a compound, which is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds described herein are unsolvated. Inother embodiments, one or more of the compounds are in solvated form. Asknown in the art, the solvate can be any of pharmaceutically acceptablesolvent, such as water, ethanol, and the like.

Methods

HDAC6 (Histone Deacetylase 6) inhibitors have shown efficacy in animalmodels of hematologic cancers and CNS disorders. Provided herein is ause of HDAC6 inhibitors for the treatment or prevention of diabeticperipheral neuropathy.

As used herein, and unless otherwise specified, the term “DiabeticPeripheral Neuropathy” (DPN), also called diabetic neuropathy, DN or“Diabetic Peripheral Neuropathic Pain” (DPNP), refers to chronic paincaused by neuropathy associated with diabetes mellitus. The classicpresentation of DPN is pain or tingling in the feet that can bedescribed not only as “burning” or “shooting” but also as severe achingpain. Less commonly, patients may describe the pain as itching, tearing,or like a toothache. The pain may be accompanied by allodynia andhyperalgesia and an absence of symptoms, such as numbness.

Compounds 1, 2, and 3 have been studied in models of DPN (see, e.g.,Example 5). Compound 2 has been studied in in vitro and in vivo modelsof DPN (see Examples 4 and 5).

In one aspect, provided herein are methods of treating or preventing adiabetic peripheral neuropathy in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of anHDAC6 inhibitor.

In an embodiment of the method, the HDAC6 inhibitor is an HDAC6-specificinhibitor.

In an embodiment of the method, the HDAC6 inhibitor is a compound ofFormula Ia, or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of Formula Ia is:

or a pharmaceutically acceptable salt thereof.

In an embodiment of the method, the HDAC6 inhibitor is a compound ofFormula I, or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of Formula I is:

or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of Formula I is:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the HDAC6 inhibitor is a compound of Formula II,or a pharmaceutically acceptable salt thereof.

In a specific embodiment, the compound of Formula II is:

or a pharmaceutically acceptable salt thereof.

In another specific embodiment, the compound of Formula II is:

or a pharmaceutically acceptable salt thereof.

For any of the above methods or uses, the required dosage will varydepending on the mode of administration, the particular condition to betreated and the effect desired.

According to the methods of treatment of the present invention,disorders are treated or prevented in a subject, such as a human orother animal, by administering to the subject a therapeuticallyeffective amount of a compound of the invention, in such amounts and forsuch time as is necessary to achieve the desired result. The term“therapeutically effective amount” of a compound of the invention meansa sufficient amount of the compound so as to decrease the symptoms of adisorder in a subject. As is well understood in the medical arts atherapeutically effective amount of a compound of this invention will beat a reasonable benefit/risk ratio applicable to any medical treatment.

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount may vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors.

In certain embodiments, a therapeutic amount or dose of the compounds ofthe present invention may range from about 0.1 mg/kg to about 500 mg/kg(about 0.18 mg/m² to about 900 mg/m²), alternatively from about 1 toabout 50 mg/kg (about 1.8 to about 90 mg/m²). In general, treatmentregimens according to the present invention comprise administration to apatient in need of such treatment from about 10 mg to about 1000 mg(including e.g., about 10 mg to 500 mg) of the compound(s) of thisinvention per day in single or multiple doses. Thus, in an embodiment ofthe methods of treatment provided herein, the compound according to thepresent invention is administered at a dosage of 10 mg, 20 mg, 30 mg, 40mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg,140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg,230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg,320 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg,420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, or 500mg per day. In a further embodiment, the compound according to thepresent invention is administered at a dosage of 20 mg, 40 mg, 60 mg, 80mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, or 200 mg per day. In yetanother embodiment, the the compound according to the present inventionis administered at a dosage of 80 or 120 mg per day. In an embodiment,the compound according to the present invention is administered one timedaily. Therapeutic amounts or doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

Use

Also provided herein is an HDAC6 inhibitor for use in the treatment orprevention of diabetic peripheral neuropathy in a subject. In anembodiment, the HDAC6 inhibitor is an HDAC6-specific inhibitor.

In a further embodiment, HDAC6 inhibitor is a compound of Formula Ia, ora pharmaceutically acceptable salt thereof. In yet a further embodiment,the HDAC inhibitor is a compound of Formula I, or a pharmaceuticallyacceptable salt thereof. The compound of Formulae Ia or I can beselected from the group consisting of:

or pharmaceutically acceptable salt thereof.

In another embodiment, the HDAC6 inhibitor is a compound of Formula II,or a pharmaceutically acceptable salt thereof.

In an embodiment, the compound of Formula II is:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the compound of Formula II is:

or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a use of a compound selected from thegroup consisting of:

or pharmaceutically acceptable salts thereof, for the preparation of amedicament for the treatment or prevention of diabetic peripheralneuropathy.

In an embodiment of this use, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment of this use, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment of this use, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment of this use, the compound is

or a pharmaceutically acceptable salt thereof.

In another embodiment of this use, the compound is

or a pharmaceutically acceptable salt thereof.Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical compositioncomprising any of the compounds provided herein, or pharmaceuticallyacceptable salts thereof, together with a pharmaceutically acceptablecarrier for use in the treatment or prevention of diabetic peripheralneuropathy.

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of a compound of the present inventionformulated together with one or more pharmaceutically acceptablecarriers. The term “pharmaceutically acceptable carrier” means anon-toxic, inert solid, semi-solid or liquid filler, diluent,encapsulating material or formulation auxiliary of any type. Thepharmaceutical compositions of this invention can be administered tohumans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, or as an oral or nasal spray.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, forexample, orally, e.g., in the form of tablets or capsules, orparenterally, e.g., in the form of injectable solutions or suspensions,topically, e.g., in the form of lotions, gels, ointments or creams, orin a nasal or suppository form. Pharmaceutical compositions comprising acompound of the present invention in free form or in a pharmaceuticallyacceptable salt form in association with at least one pharmaceuticallyacceptable carrier or diluent can be manufactured in a conventionalmanner by mixing, granulating or coating methods. For example, oralcompositions can be tablets or gelatin capsules comprising the activeingredient together with a) diluents, e.g., lactose, dextrose, sucrose,mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g.,silica, talcum, stearic acid, its magnesium or calcium salt and/orpolyethyleneglycol; for tablets also c) binders, e.g., magnesiumaluminum silicate, starch paste, gelatin, tragacanth, methylcellulose,sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions may be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations may also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such maycontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties. Unless otherwise defined, alltechnical and scientific terms used herein are accorded the meaningcommonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

EXAMPLES

Examples have been set forth below for the purpose of illustration andto describe certain specific embodiments of the invention. However, thescope of the claims is not to be in any way limited by the examples setforth herein. Various changes and modifications to the disclosedembodiments will be apparent to those skilled in the art and suchchanges and modifications including, without limitation, those relatingto the chemical structures, substitutents, derivatives, formulationsand/or methods of the invention may be made without departing from thespirit of the invention and the scope of the appended claims.

Example 1: Synthesis of Compounds of Formulae Ia and I A. Synthesis ofN-hydroxy-2-((1-phenylcyclopropyl)amino)pyrimidine-5-carboxamide(Compound 1)

Synthesis of Intermediate 2

A solution of intermediate 1, benzonitrile, (250 g, 1.0 equiv.), andTi(OiPr)₄ (1330 ml, 1.5 equiv.) in MBTE (3750 ml) was cooled to about−10 to −5° C. under a nitrogen atmosphere. EtMgBr (1610 ml, 3.0 M, 2.3equiv.) was added dropwise over a period of 60 min., during which theinner temperature of the reaction was kept below 5° C. The reactionmixture was allowed to warm to 15-20° C. for 1 hr. BF₃-ether (1300 ml,2.0 equiv.) was added dropwise over a period of 60 min., while the innertemperature was maintained below 15° C. The reaction mixture was stirredat 15-20° C. for 1-2 hr. and stopped when a low level of benzonitrileremained. 1N HCl (2500 ml) was added dropwise while maintaining theinner temperature below 30° C. NaOH (20%, 3000 ml) was added dropwise tobring the pH to about 9.0, while still maintaining a temperature below30° C. The reaction mixture was extracted with MTBE (3 L×2) and EtOAc (3L×2), and the combined organic layers were dried with anhydrous Na₂SO₄and concentrated under reduced pressure (below 45° C.) to yield a redoil. MTBE (2500 ml) was added to the oil to give a clear solution, andupon bubbling with dry HCl gas, a solid precipitated. This solid wasfiltered and dried in vacuum yielding 143 g of intermediate 2.

Synthesis of Intermediate 4

Intermediate 2 (620 g, 1.0 equiv) and DIPEA (1080 g, 2.2 equiv. weredissolved in NMP (3100 ml) and stirred for 20 min. Intermediate 3 (680g, 1.02 equiv.) was added and the reaction mixture was heated to about85-95° C. for 4 hrs. The solution was allowed to slowly cool to r.t.This solution was poured onto H₂O (20 L) and much of the solid wasprecipitated out from the solution with strong stirring. The mixture wasfiltered and the cake was dried under reduced pressure at 50° C. for 24hr., yielding 896 g of intermediate 4 (solid, 86.8%).

Synthesis ofN-hydroxy-2-((1-phenylcyclopropyl)amino)pyrimidine-5-carboxamide(Compound 1)

A solution of MeOH (1000 ml) was cooled to about 0-5° C. with stirring.NH₂OH HCl (1107 g, 10 equiv.) was added, followed by careful addition ofNaOCH₃ (1000 g, 12.0 equiv.) The resulting mixture was stirred at 0-5°C. for one hour, and was filtered to remove the solid. Intermediate 4(450 g, 1.0 equiv.) was added to the reaction mixture in one portion,and stirred at 10° C. for two hours until intermediate 4 was consumed.The reaction mixture was adjusted to a pH of about 8.5-9 throughaddition of HCl (6N), resulting in precipitation. The mixture wasconcentrated under reduced pressure. Water (3000 ml) was added to theresidue with intense stirring and the precipitate was collected byfiltration. The product was dried in an oven at 45° C. overnight (340 g,79% yield).

B. Synthesis ofN-hydroxy-2-((2-methoxy-5-(trifluoromethyl)benzyl)amino)pyrimidine-5-carboxamide(Compound 3)

Synthesis of Intermediate 2

To mixture of 2-methoxy-5-(trifluoromethyl)benzonitrile (300 mg, 1.5mmol) in Dry THF (15 ml) was added LiAlH₄ (285 mg, 7.5 mmol, solid)slowly at ice temperature. Then the mixture was stirred at roomtemperature overnight. After the reaction was deemed complete from TLC,the mixture was quenched with sat. NH₄Cl and extracted with EA (1*20ml). The organic layer was dried and concentrated to afford the crudeamine intermediate 2 (300 mg, crude) as light yellow oil.

Synthesis of Intermediate 3

To a solution of crude (2-methoxy-5-(trifluoromethyl)phenyl)-methanamine(300 mg) in NMP (8 ml) was added 2-Cl-pyrimidine (272 mg, 1.0 eq) andDIPEA (943 mg, 5.0 eq). The mixture was stirred at 115° C. for 2 h. Themixture was diluted with water (10 ml), extracted with EA (1*15 ml),dried and concentrated to afford a residue, which was purified byprep-TLC with PE/EA=3:1 to afford intermediate 3 (200 mg, 37.6%, 2steps) as light yellow solid.

Synthesis ofN-hydroxy-2-((2-methoxy-5-(trifluoromethyl)benzyl)amino)pyrimidine-5-carboxamide(Compound 3)

To a mixture of ethyl2-(2-methoxy-5-(trifluoromethyl)benzylamino)pyrimidine-5-carboxylate(100 mg, 0.28 mmol) in MeOH/DCM (6/2 ml) was added NH₂OH (0.5 ml),followed NaOH solution (sat. in MeOH, 1 ml) dropwise at 0° C. Themixture was stirred at 0° C. for 3 hrs. After the reaction was deemedcomplete from TLC, the mixture was concentrated to remove MeOH and DCM,acidified to pH near 6-7, and then purified by Prep-HPLC to afford thedesired product, Compound 3, as white solid (30 mg, 31%). LCMS: m/z=343(M+H)⁺. ¹H NMR (400 MHz, DMSO) δ 11.03 (s, 1H), 8.99 (s, 1H), 8.61 (d,J=17.4 Hz, 2H), 8.22 (t, J=6.2 Hz, 1H), 7.61 (d, J=8.5 Hz, 1H), 7.42 (d,J=1.8 Hz, 1H), 7.19 (d, J=8.6 Hz, 1H), 4.55 (d, J=6.1 Hz, 2H), 3.91 (s,3H).

Example 2: Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound 2)

Step 1: Synthesis of Intermediate 2

A mixture of aniline (3.7 g, 40 mmol), ethyl2-chloropyrimidine-5-carboxylate 1 (7.5 g, 40 mmol), K₂CO₃ (11 g, 80mmol) in DMF (100 ml) was degassed and stirred at 120° C. under N₂overnight. The reaction mixture was cooled to rt and diluted with EtOAc(200 ml), then washed with saturated brine (200 ml×3). The organic layerwas separated and dried over Na₂SO₄, evaporated to dryness and purifiedby silica gel chromatography (petroleum ethers/EtOAc=10/1) to give thedesired product as a white solid (6.2 g, 64%).

Step 2: Synthesis of Intermediate 3

A mixture of intermediate 2 (6.2 g, 25 mmol), iodobenzene (6.12 g, 30mmol), CuI (955 mg, 5.0 mmol), Cs₂CO₃ (16.3 g, 50 mmol) in TEOS (200 ml)was degassed and purged with nitrogen. The resulting mixture was stirredat 140° C. for 14 h. After cooling to r.t., the residue was diluted withEtOAc (200 ml) and 95% EtOH (200 ml), NH₄F—H₂O on silica gel [50 g,pre-prepared by the addition of NH₄F (100 g) in water (1500 ml) tosilica gel (500 g, 100-200 mesh)] was added, and the resulting mixturewas kept at r.t. for 2 h, the solidified materials was filtered andwashed with EtOAc. The filtrate was evaporated to dryness and theresidue was purified by silica gel chromatography (petroleumethers/EtOAc=10/1) to give a yellow solid (3 g, 38%).

Step 3: Synthesis of Intermediate 4

2N NaOH (200 ml) was added to a solution of intermediate 3 (3.0 g, 9.4mmol) in EtOH (200 ml). The mixture was stirred at 60° C. for 30 min.After evaporation of the solvent, the solution was neutralized with 2NHCl to give a white precipitate. The suspension was extracted with EtOAc(2×200 ml), and the organic layer was separated, washed with water(2×100 ml), brine (2×100 ml), and dried over Na₂SO₄. Removal of solventgave a brown solid (2.5 g, 92%).

Step 4: Synthesis of Intermediate 6

A mixture of intermediate 4 (2.5 g, 8.58 mmol), aminoheptanoate 5 (2.52g, 12.87 mmol), HATU (3.91 g, 10.30 mmol), DIPEA (4.43 g, 34.32 mmol)was stirred at r.t. overnight. After the reaction mixture was filtered,the filtrate was evaporated to dryness and the residue was purified bysilica gel chromatography (petroleum ethers/EtOAc=2/1) to give a brownsolid (2 g, 54%).

Step 5: Synthesis of2-(diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide(Compound 2)

A mixture of intermediate 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0° C. for 10 min.Hydroxylamine (50%) (10 ml) was cooled to 0° C. and added to themixture. The resulting mixture was stirred at r.t. for 20 min. Afterremoval of the solvent, the mixture was neutralized with 1M HCl to givea white precipitate. The crude product was filtered and purified bypre-HPLC to give a white solid (950 mg, 48%).

Example 3: HDAC Enzyme Assays

Compounds for testing were diluted in DMSO to 50 fold the finalconcentration and a ten point three fold dilution series was made. Thecompounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl,0.001% Tween-20, 0.05% BSA, 20 μM TCEP) to 6 fold their finalconcentration. The HDAC enzymes (purchased from BPS Biosciences) werediluted to 1.5 fold their final concentration in assay buffer. Thetripeptide substrate and trypsin at 0.05 μM final concentration werediluted in assay buffer at 6 fold their final concentration. The finalenzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The finalsubstrate concentrations used were 16 μM (HDAC1), 10 μM (HDAC2), 17 μM(HDAC3) and 14 μM (HDAC6). Five 1 of compound and 20 μl of enzyme wereadded to wells of a black, opaque 384 well plate in duplicate. Enzymeand compound were incubated together at room temperature for 10 minutes.Five μl of substrate was added to each well, the plate was shaken for 60seconds and placed into a Victor 2 microtiter plate reader. Thedevelopment of fluorescence was monitored for 60 min and the linear rateof the reaction was calculated. The IC₅₀ was determined using Graph PadPrism by a four parameter curve fit. The IC₅₀ values for Compounds 1, 2,and 3 are shown below in Table 1.

TABLE 1 HDAC1 HDAC2 HDAC3 HDAC6 Compound IC50 (nM) IC50 (nM) IC50 (nM)IC50 (nM) Compound 1 94 128 158 1.7 Compound 2 58 64 84 10 Compound 3 6882 105 2

Example 4: HDAC6 Inhibition Restores Axonal Transport Impaired by HighGlucose (Hyperglycemia) in Cultured Neurons

Microtubule-based axonal transport plays a pivotal role in normalneuronal function. Deficits in axonal transport have been reported inSTZ rats and axonal transport abnormalities are thought to contribute toperipheral nerve dysfunction and degeneration. HDAC6 is a tubulindeacetylase and HDAC6 inhibition has been shown to restore normal axonaltransport in different models of neurodegenerative disorders andperipheral neuropathies.

To investigate the effects of hyperglycemia on axonal transport liveimaging was used to measure mitochondrial transport in primary ratdorsal root ganglion cells. Primary rat dorsal root ganglion (DRG)neurons (Lonza) were cultured for 5-7 days according to the vendor'sprotocol. The day before the imaging session cells were infected usingCellLight Mitochondria-GFP (BacMam 2.0-based reagent, ThermoFisherScientific) to fluorescently label mitochondria. Additionally, the mediawas replaced with media containing 200 mM glucose or 200 mM glucose and500 nM Compound 2. Media in control wells was replaced with fresh mediacontaining 25 mM glucose (control). Cells were allowed to incubate underthese conditions for 24 h. For live imaging cells were maintained inCO2-independent media, containing either 25 mM glucose, 200 mM glucoseor 200 mM glucose and 500 mM Compound 2. Imaging was performed usingZeiss 3i system (Intelligent Imaging Innovations) in temperatecontrolled environment (37 C). Mitochondria were visualized using FITCfilter. Time lapse images were acquired every 2 seconds over the courseof 2 min and analyzed using Fiji (ImageJ). MtrackJ plugin was used tomanually track mitochondrial movements and calculate parameters such astotal distance traveled by each mitochondria. Total distance traveled bya population of mitochondria in each condition was plotted as acumulative frequency plot. Data presented is a pool of 5 independentexperiments.

Neurons exposed to high glucose exhibit mitochondrial transport deficitsas shown by the shift in the cumulative frequency plot (FIG. 1A) and adecrease in the mean value of the total distance traveled (FIG. 1B).Treatment with Compound 2 restores normal pattern of mitochondrialmovement.

This study shows that neurons exposed to high glucose exhibit axonaltransport deficits and that treatment with Compound 2 rescues thesedeficits (see FIG. 1A). Thus, Compound 2 can be used as an effectivetreatment option for diabetic peripheral neuropathy.

Example 5: HDAC6 Inhibition Reverses Neuropathic Pain in STZ DiabeticRats

Compounds 1, 2, and 3 were tested for the ability to reverse neuropathyin streptozotocin-induced diabetic rats (STZ rats), a commonly usedmodel of painful DPN. Both compounds reversed tactile allodynia in STZrats in a dose-dependent manner and restored the pain threshold in theserats to nearly normal levels. In the case of Compound 1, e.g., thepositive effect of the drug on pain threshold persisted even after thedrug treatment was stopped, suggesting a disease modifying effect ofHDAC6 inhibitors in DPN.

Further, Compound 1 penetrates the blood brain barrier, while Compound 2does not penetrate the blood brain barrier very well.

Diabetes was induced in male SD rats by IV dosing of STZ (60 mg/kg) onstudy days 0-10. Blood glucose level (BGL) of above 300 mg/dl wasobserved on study day 3, following STZ dosing, which maintainedthroughout the study period. On study day 10, the sensitivity of allanimals to von Frey filaments was tested and animals that showed adecrease in withdrawal force threshold (average pain threshold of <43 gfor both hind paws) were included in the study and assigned to thetreatment groups.

Animals were dosed as indicated twice daily for 8 days (e.g., Compound 2orally at 3, 10, and 30 mg/kg twice daily (BID) on days 11-21 andCompound 1 orally at 1, 3, and 10 mg/kg once daily (QD) on days 11-21).Gabapentin (150 mg/kg) was used as a positive control. Tactile allodyniawas measured on the indicated days during dosing and once on day 24,three days after dosing was stopped. The results of the von Frey testupon dosing with Compound 1 are shown in FIG. 2A. The results of the vonFrey test upon dosing with Compound 2 are shown in FIG. 2B. The resultsof the von Frey test upon dosing with Compound 3 are shown in FIG. 2C.Vehicle treated animals experienced low mechanical threshold throughoutthe treatment period (days 11-21) and also 3 days after the last dose(day 24). Compounds 1, 2, and 3 reversed tactile allodynia in STZ ratsin a dose dependent manner and restored the pain threshold in these ratsto almost normal levels (see FIGS. 2A, 2B, and 2C). Further, the effectof Compound 1 persisted even after the drug was withdrawn (FIG. 2A, day24).

Example 6: Compound 2 Increases Tubulin Acetylation in Neurons

Dorsal root ganglion neurons (DRGNs) were treated with Compound 2 for 24hours and whole cell extracts were analyzed by western blot. Compound 2induced an increase in tubulin acetylation in a dose-dependent matter.Minimal histone acetylation increase is observed in DRGNs in response toCompound 2 at up to 1 uM. Compound 2 induces tubulin hyperacetylation inwhole cell lysates of rat DRGNs (see FIG. 3A).

DRGNs were treated with Compound 2 on coverslips, fixed and stained withantibodies against acetyl-tubulin and total tubulin (fluorescent ICC) tovisualize and quantitate tubulin acetylation in axonal microtubules.Compound 2 increases acetylation of axonal microtubules (See FIG. 3B).Acetyl-tubulin signal was normalized to total tubulin signal.

Example 7: HDAC6 Inhibitors do not Act as General Analgesics

A Tail Flick test was performed every hour in a time period of fourhours after treatments. Rats were given Compound 1 or Compound 2 eitherfor 4 days prior to the Tail Flick test (designated as BID groups) or ina single dose (QD groups) 1 h before the test.

Data are presented in FIG. 4 as the latency time in seconds until theanimals flicked their tails in response to the heat source. Morphine andGabapentin were used as positive controls. Treatments with Compound 1and Compound 2 were not effective (except in the group that receivedCompound 2 at 30 mg/kg BID for 4 days) in increasing latency time whencompared to vehicle control group

Example 8: Metabolomic Study of HDAC6 Inhibitor Compound 1 in DiabeticNeuropathic Pain Model in Rats

Metabolic stress underlies much of the pathology associated withdiabetes. Freeman et al. (Freeman, O. J., Unwin, R. D., Dowsey, A. W.,Begley, P., Ali, S., Hollywood, K. A., Rustogi, N., Petersen, R. S.,Dunn, W. B., Cooper, G. J. S., Gardiner, N. J. (2016) “MetabolicDysfunction is Restricted to the Sciatic Nerve in Experimental DiabeticNeuropathy” Diabetes 65) showed that in rats treated with streptozotocinto induce hyperglycemia, changes in metabolites in the sciatic nervewere associated with an increase in neuropathic pain.

Hyperglycemia was induced in rats with an injection of streptozotocin(60 mg/kg). Ten days later neuropathic pain was confirmed using a vonFrey filament test for tactile allodynia. In this test, filaments ofincreasing diameter were applied to the plantar surface of the rear pawand the force necessary to precipitate a paw withdrawal is determined bythe smallest filament needed to invoke a response. After tactileallodynia was confirmed the animals were dosed with Compound 1 twice perday at 10 mg/kg. At day 21 (11 days after the start of dosing) threeeach of vehicle and Compound 1 treated animals were sacrificed and thedorsal root ganglia and sciatic nerves were dissected from both sidesand snap frozen. In addition, three naïve rats and three rats that hadtactile allodynia at day 10 were sacrificed and the dorsal root gangliaand sciatic nerves were dissected and frozen as control samples.

Dorsal root ganglia and sciatic nerves were sent to Human MetabolomeTechnologies for analysis of metabolic intermediates. Extracts fromthese samples were analyzed by capillary electrophoresis/massspectrometry to measure the level of charged metabolites. In addition,sciatic nerve extracts were analyzed by liquid chromatography/massspectrometry to measure the level of non-charged metabolites.

Overall, 314 different metabolites were identified and 81 metabolitesquantitated in sciatic nerve samples. 242 metabolites were identifiedand 83 quantitated in dorsal root ganglion samples. There were clearquantitative differences in metabolites in diabetic as compared to naïverats in both tissues. As an example, the diabetic animals showed a largeincrease in sorbitol in the dorsal root ganglion tissues, which wasreported in Freeman et al. (2016) for a similar animal model. In thesciatic nerve, diabetic animals showed a sharp decrease in the level oflysine, which was reported in Freeman et al. as well.

The most dramatic changes in metabolites described in the Freeman paperoccurred among the oxidized fatty acids, including acyl-carnitines. Inour study the diabetic rats had a significant increase in severalacyl-carnitines, including AC 16:2 (FIG. 5A) and AC 18:2 (FIG. 5B).Treatment with Compound 1 for 11 days normalized many of the metabolicchanges, including the changes in acyl-carnitines. This reversal ofacyl-carnitine buildup may represent one mechanism of neuroprotection byHDAC6 inhibition, since acyl-carnitine production in peripheral nervesis associated with axonal degeneration (Viader, A., Sasaki, Y., Kim, S.,Strickland, A., Workman, C. S., Yang, K., Gross, R. W., Milbrandt, J.(2014) “Abberant Schwann Cell Lipid Metabolism Linked to MitochondrialDeficits Leads to Axon Degeneration and Neuropathy” Neuron 77(5)).

In view of the Examples presented herein, HDAC6 inhibitors of FormulaeIa, I, and II, e.g., Compounds 1, 2, and 3 can be used for the treatmentof DPN.

The invention claimed is:
 1. A method for treating diabetic peripheralneuropathy in a subject in need thereof comprising administering to thesubject a therapeutically effective amount of an HDAC6-specificinhibitor.
 2. The method of claim 1, wherein the HDAC6-specificinhibitor is a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein ring B is aryl orheteroaryl optionally substituted by OH, halo, or C₁₋₆-alkyl; R₁ is arylor heteroaryl optionally substituted by OH, halo, or C₁₋₆-alkyl; and Ris H or C₁₋₆-alkyl.
 3. The method of claim 2, wherein the compound ofFormula II is:

or a pharmaceutically acceptable salt thereof.
 4. The method of claim 1,wherein the HDAC6-specific inhibitor is administered by oraladministration.